Leica application suite v3.6

Sections from wt and ZnT2ko mice (n = 4–6 mice/genotype) were used for IF staining. Antibodies used were as follows: anti–8-hydroxydeoxyguanosine (20 μg/mL, ab62623; Abcam, Cambridge, MA), ZnT2 (4 μg/mL, sc-27507; Santa Cruz Antibody, Santa Cruz, CA), Reg-IIIγ (1:100; a gift from Dr Matam Vijay-Kumar), Bmi-1 (12 μg/mL, ab38295; Abcam), and Lgr-5 (1:25, ab75732; Abcam). Primary antibodies were visualized with donkey anti-goat IgG Alexa Fluor 488 (4 μg/mL, A11055; Life Technologies, Frederick, MD), donkey anti-rabbit IgG Alexa Fluor 594 (4 μg/mL, A21207; Life Technologies), or anti-mouse IgG Alexa Fluor 488 (4 μg/mL, A21202; Life Technologies), and counterstained with 4’,6-diamidino-2-phenylindole (175 μg/mL; Life Technologies). Images were collected at 20×, 40×, or under oil at 63× magnification using a Leica DM IL LED microscope with a Leica DFC425 digital camera (Leica Microsystems, Buffalo Grove, IL) or using a Leica Inverted Confocal Microscope SP8 (Leica Microsystems). Images were collected using Leica Application Suite (V3.6) and saved as .tiff files. Brightness was adjusted uniformly across all images using Adobe Photoshop CS3 version 10.0.

The terminal ileum (n = 4/genotype) was frozen in tissue-freezing medium (Tissue-Tek OCT; Fisher Scientific) floated in an isopropanol/dry ice bath. Sections (10 μm) were cut using a CM 1950 cryostat (Leica), and the fluorescent reporter Zinpyr-1 (ZP1; Toronto Research Chemicals, North York, Ontario) was used to detect labile Zn pools within secretory granules as previously described.26 (link), 37 (link) Images were obtained at 40× magnification using Leica Application Suite (V3.6) on a Leica DM IL LED microscope with a Leica DFC425 digital camera. Brightness was adjusted uniformly across all images using Adobe Photoshop CS3 version 10.0.